U.S. patent application number 17/628061 was filed with the patent office on 2022-08-18 for inhibitors and use thereof in cancer treatment.
The applicant listed for this patent is The University of Sydney. Invention is credited to Robert Baxter.
Application Number | 20220259265 17/628061 |
Document ID | / |
Family ID | 1000006360359 |
Filed Date | 2022-08-18 |
United States Patent
Application |
20220259265 |
Kind Code |
A1 |
Baxter; Robert |
August 18, 2022 |
Inhibitors and Use Thereof in Cancer Treatment
Abstract
The invention generally relates to inhibitors of DNA double
strand break (DSB) repair in cancer cells exposed to DNA-damaging
chemotherapy drugs or radiotherapy. In particular, agents that
inhibit binding between insulin-like growth factor binding
protein-3 (IGFBP-3) and non-POU (pituitary-specific Pit-1,
octamer-binding proteins Oct-1 and Oct-2, and neural Unc-86)
domain-containing octamer-binding protein (NONO) and methods of
using such agents to enhance chemosensitivity or radiosensitivity
in cancer treatment are disclosed.
Inventors: |
Baxter; Robert; (St
Leonards, New South Wales, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Sydney |
Sydney, New South Wales |
|
AU |
|
|
Family ID: |
1000006360359 |
Appl. No.: |
17/628061 |
Filed: |
July 30, 2020 |
PCT Filed: |
July 30, 2020 |
PCT NO: |
PCT/AU2020/050779 |
371 Date: |
January 18, 2022 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/7048 20130101;
A61P 35/00 20180101; A61K 31/502 20130101; A61K 38/00 20130101;
C07K 7/08 20130101; A61K 31/4184 20130101 |
International
Class: |
C07K 7/08 20060101
C07K007/08; A61K 31/7048 20060101 A61K031/7048; A61K 31/502
20060101 A61K031/502; A61K 31/4184 20060101 A61K031/4184; A61P
35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 30, 2019 |
AU |
2019902712 |
Claims
1. An agent that inhibits the interaction between IGFBP-3 and
NONO.
2. An isolated peptide comprising residues:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9,
wherein X.sub.1 is His, X.sub.2 is Leu, X.sub.3 is Lys, X.sub.4 is
Phe, X.sub.5 is Leu, X.sub.6 is Asn, X.sub.7 is Val, X.sub.8 is Leu
and X.sub.9 is Ser, or conservative substitutions thereof, or a
pharmaceutically acceptable salt of the peptide.
3. The peptide of claim 2, wherein the peptide comprises the
sequence: His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser-Pro-Arg-Gly, or
conservative substitutions thereof, or a pharmaceutically
acceptable salt of the peptide.
4. The peptide of claim 2, wherein the peptide comprises the
sequence: Thr-Leu-Asn-His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser, or
conservative substitutions thereof, or a pharmaceutically
acceptable salt thereof.
5. (canceled)
6. (canceled)
7. (canceled)
8. A pharmaceutical composition comprising an agent of claim 1, and
optionally at least one pharmaceutically acceptable excipient.
9. The pharmaceutical composition of claim 8 further comprising a
chemotherapeutic agent, a radiomimetic agent or a PARP
inhibitor.
10. The pharmaceutical composition of claim 9, wherein the
chemotherapeutic agent is selected from the group consisting of a
bifunctional alkylator, a monofunctional alkylator, a topoisomerase
inhibitor, an antimetabolite, a replication inhibitor and a
platinum drug.
11. The pharmaceutical composition of claim 10, wherein the
chemotherapeutic agent is etoposide.
12. The pharmaceutical composition of claim 9, wherein the PARP
inhibitor is selected from the group consisting of veliparib and
olaparib.
13. A pharmaceutical composition comprising an isolated peptide of
claim 2, and optionally at least one pharmaceutically acceptable
excipient.
14. The pharmaceutical composition of claim 13 further comprising a
chemotherapeutic agent, a radiomimetic agent or a PARP
inhibitor.
15. The pharmaceutical composition of claim 14, wherein the
chemotherapeutic agent is selected from the group consisting of a
bifunctional alkylator, a monofunctional alkylator, a topoisomerase
inhibitor, an antimetabolite, a replication inhibitor and a
platinum drug.
16. The pharmaceutical composition of claim 15, wherein the
chemotherapeutic agent is etoposide.
17. The pharmaceutical composition of claim 14, wherein the PARP
inhibitor is selected from the group consisting of veliparib and
olaparib.
18. A method of enhancing chemosensitivity or radiosensitivity in
cancer treatment comprising administering to a subject in need
thereof a therapeutically effective amount of an agent of claim 1,
wherein the cancer is an IGFBP-3 expressing cancer.
19. The method of claim 18, wherein the IGFBP-3 expressing cancer
is breast cancer, prostate cancer, pancreatic cancer, glioblastoma
cancer or Triple Negative Breast Cancer (TNBC).
20. A method of enhancing chemosensitivity or radiosensitivity in
cancer treatment comprising administering to a subject in need
thereof a therapeutically effective amount of an isolated peptide
of claim 2, wherein the cancer is an IGFBP-3 expressing cancer.
21. The method of claim 20, wherein the IGFBP-3 expressing cancer
is breast cancer, prostate cancer, pancreatic cancer, glioblastoma
cancer or Triple Negative Breast Cancer (TNBC).
22. A method of enhancing chemosensitivity or radiosensitivity in
TNBC treatment comprising administering to a subject in need
thereof a therapeutically effective amount of an agent of claim
1.
23. A method of enhancing chemosensitivity or radiosensitivity in
TNBC treatment comprising administering to a subject in need
thereof an isolated peptide of claim 2.
Description
FIELD OF THE INVENTION
[0001] The present invention generally relates to inhibitors of DNA
double-strand break (DSB) repair. In particular, the present
invention relates to agents that inhibit binding between
insulin-like growth factor binding protein-3 (IGFBP-3) and non-POU
(pituitary-specific Pit-1, octamer-binding proteins Oct-1 and
Oct-2, and neural Unc-86) domain-containing octamer-binding protein
(NONO), and methods of using such agents to enhance
chemosensitivity or radiosensitivity in cancer treatment.
BACKGROUND
[0002] Any discussion of the prior art throughout the specification
should in no way be considered as an admission that such prior art
is widely known or forms part of common general knowledge in the
field.
[0003] The mechanism of action of many chemo- and radiotherapies is
the induction of DSB in cancer cell DNA. In response, cancer cells
can either enter a program of cell death or DSB repair. A common
DSB repair mechanism is non-homologous end-joining (NHEJ). This
pathway is referred to as "non-homologous" because unlike the other
classic DSB repair mechanism, homologous recombination (HR), NHEJ
does not require a homologous template for repair of the DNA
lesion. As DNA damage repair makes chemo- and radiotherapy less
effective, agents that inhibit DNA repair pathways enhance the
specificity and effectiveness of chemo- and radiotherapy and may
help overcome cancer treatment resistance.
[0004] Triple Negative Breast Cancers (TNBC) are unresponsive to
estrogen receptor or human epidermal growth factor receptor 2
(HER2) directed treatments. TNBC is a more aggressive form of
breast cancer with a high prevalence in younger women and is
associated with an unfavorable prognosis. There has been limited
therapeutic progress for treating TNBC in the past several decades
and cytotoxic chemotherapy is still the standard of care. However,
their responsiveness may be blunted by DNA DSB repair. There is
thus an urgent unmet need to develop effective agents for
sensitizing DNA-damaging chemotherapy drugs or radiotherapy,
especially for this patient population.
[0005] It is an object of the present invention to overcome or
ameliorate at least one of the disadvantages of the prior art, or
to provide a useful alternative.
SUMMARY OF THE INVENTION
[0006] Chemotherapies and radiotherapies induce DNA DSB in cancer
cell DNA. Cancer cells in turn can either enter a program of cell
death or DNA damage repair. An important pathway for DNA DSB repair
is NHEJ. The protein IGFBP-3 is involved in DSB repair by NHEJ and
the inventor has unexpectedly found that DNA- and RNA-binding
protein NONO (and its dimerization partner splicing factor,
proline/glutamine-rich (SFPQ)) interacts with IGFBP-3 in TNBC cell
lines exposed to chemotherapy drugs and promotes DNA DSB
repair.
[0007] The invention generally relates to inhibitors of DNA DSB
repair in cancer cells exposed to DNA-damaging chemotherapy drugs
or radiotherapy. In particular, the present invention relates to
agents that inhibit binding between IGFBP-3 and NONO, and methods
of using such agents to enhance chemosensitivity or
radiosensitivity in cancer treatment.
[0008] Provided are agents that inhibit the interaction between
IGFBP-3 and NONO and inhibit DNA DSB repair. Such agents enhance
chemosensitivity or radiosensitivity. The agents as described
herein may be a small molecule, substance or compound that inhibits
the interaction between IGFBP-3 and NONO and thus inhibits DNA DSB
repair following chemotherapy or radiotherapy.
[0009] Provided are isolated peptides that inhibit the interaction
between NONO and IGFBP-3. The peptides of the invention may be
derived from the full length sequence of mature human IGFBP-3.
[0010] Provided are methods for enhancing chemosensitivity or
radiosensitivity in cancer treatment comprising administering to a
subject in need thereof an agent that inhibits the interaction
between NONO and IGFBP-3. In particular, provided are methods for
enhancing chemosensitivity or radiosensitivity in TNBC treatment.
Agents that inhibit the interaction between NONO and IGFBP-3 may be
suitable for neoadjuvant or adjuvant therapy to be used in
conjunction with radiotherapies or other chemotherapeutic
approaches.
[0011] Provided is a therapy for enhancing chemosensitivity or
radiosensitivity in cancer treatment comprising administering an
agent that inhibits the interaction between NONO and IGFBP-3.
Exemplary cancers that may be treated include, but are not limited
to, breast cancer, prostate cancer, pancreatic cancer, glioblastoma
and the like. In particular, provided is a therapy for enhancing
chemosensitivity or radiosensitivity in TNBC treatment.
[0012] In a first aspect, the invention provides an agent that
inhibits the interaction between IGFBP-3 and NONO.
[0013] In a second aspect, the invention provides an isolated
peptide comprising residues:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9,
[0014] wherein X.sub.1 is His, X.sub.2 is Leu, X.sub.3 is Lys,
X.sub.4 is Phe, X.sub.5 is Leu, X.sub.6 is Asn, X.sub.7 is Val,
X.sub.8 is Leu and X.sub.9 is Ser, or conservative substitutions
thereof,
[0015] or a pharmaceutically acceptable salt of the peptide.
[0016] In certain embodiments, the peptide comprises the
sequence:
His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser-Pro-Arg-Gly,
[0017] or conservative substitutions thereof,
[0018] or a pharmaceutically acceptable salt of the peptide.
[0019] In some embodiments, the peptide comprises the sequence:
Thr-Leu-Asn-His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser,
[0020] or conservative substitutions thereof,
[0021] or a pharmaceutically acceptable salt of the peptide.
[0022] In a third aspect, the invention provides an isolated
peptide comprising residues:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9,
[0023] wherein X.sub.1 is His, X.sub.2 is Leu, X.sub.3 is Lys,
X.sub.4 is Phe, X.sub.5 is Leu, X.sub.6 is Asn, X.sub.7 is Val,
X.sub.8 is Leu and X.sub.9 is Ser, or conservative substitutions
thereof, [0024] or a pharmaceutically acceptable salt of the
peptide, wherein the peptide inhibits the interaction between
IGFBP-3 and NONO.
[0025] In certain embodiments, the peptide comprises the
sequence:
His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser-Pro-Arg-Gly,
[0026] or conservative substitutions thereof, [0027] or a
pharmaceutically acceptable salt of the peptide, wherein the
peptide inhibits the interaction between IGFBP-3 and NONO.
[0028] In some embodiments, the peptide comprises the sequence:
Thr-Leu-Asn-His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser,
[0029] or conservative substitutions thereof, [0030] or a
pharmaceutically acceptable salt of the peptide, wherein the
peptide inhibits the interaction between IGFBP-3 and NONO.
[0031] In a fourth aspect, the invention provides a pharmaceutical
composition comprising an agent of the invention, or an isolated
peptide of the invention and optionally at least one
pharmaceutically acceptable excipient. In some embodiments, the
pharmaceutical composition further comprises a chemotherapeutic
agent, a radiomimetic agent or a PARP inhibitor. In a related
embodiment, the chemotherapeutic agent is selected from the group
consisting of a bifunctional alkylator, a monofunctional alkylator,
a topoisomerase inhibitor, an antimetabolite, a replication
inhibitor and a platinum drug. In some embodiments, the
chemotherapeutic agent is etoposide. In certain embodiments, the
PARP inhibitor is veliparib. In some embodiments, the PARP
inhibitor is olaparib.
[0032] In a fifth aspect, the invention provides a method of
enhancing chemosensitivity or radiosensitivity in cancer treatment
comprising administering to a subject in need thereof a
therapeutically effective amount of an agent of the invention, an
isolated peptide of the invention or a pharmaceutical composition
of the invention, wherein the cancer is an IGFBP-3 expressing
cancer. In some embodiments, the IGFBP-3 expressing cancer is
breast cancer, prostate cancer, pancreatic cancer or glioblastoma
cancer. In certain embodiments, the IGFBP-3 expressing cancer is
Triple Negative Breast Cancer (TNBC).
[0033] In a sixth aspect, the invention provides a method of
enhancing chemosensitivity or radiosensitivity in TNBC treatment
comprising administering to a subject in need thereof a
therapeutically effective amount of an agent of the invention, an
isolated peptide of the invention or a pharmaceutical composition
of the invention.
[0034] In a seventh aspect, the invention provides use of an agent
of the invention, or an isolated peptide of the invention in the
manufacture of a medicament for enhancing chemosensitivity or
radiosensitivity in cancer treatment, wherein the cancer is an
IGFBP-3 expressing cancer.
[0035] In an eighth aspect, the invention provides use of an agent
of the invention, or an isolated peptide of the invention in the
manufacture of a medicament for enhancing chemosensitivity or
radiosensitivity in TNBC treatment.
[0036] In some embodiments, the invention provides an agent of the
invention for use in a method of enhancing chemosensitivity or
radiosensitivity in cancer treatment, wherein the cancer is an
IGFBP-3 expressing cancer. In a related embodiment, the IGFBP-3
expressing cancer is breast cancer, prostate cancer, pancreatic
cancer or glioblastoma cancer
[0037] In some embodiments, the invention provides an agent of the
invention for use in a method of enhancing chemosensitivity or
radiosensitivity in TNBC treatment.
[0038] In certain embodiments, the invention provides an isolated
peptide of the invention or a pharmaceutically acceptable salt
thereof for use in a method of enhancing chemosensitivity or
radiosensitivity in cancer treatment, wherein the cancer is an
IGFBP-3 expressing cancer. In a related embodiment, the IGFBP-3
expressing cancer is breast cancer, prostate cancer, pancreatic
cancer or glioblastoma cancer.
[0039] In some embodiments, the invention provides an isolated
peptide of the invention or a pharmaceutically acceptable salt
thereof for use in a method of enhancing chemosensitivity or
radiosensitivity in TNBC treatment.
[0040] Methods of synthesizing or generating an agent or a peptide
herein disclosed are not particularly limited and any suitable
method may be used.
[0041] Definitions
[0042] Unless the context clearly requires otherwise, throughout
the description and the claims, the words "comprise", "comprising"
and the like are to be construed in an inclusive sense as opposed
to an exclusive or exhaustive sense; that is to say, in the sense
of "including, but not limited to".
[0043] As used herein, the singular forms "a," "an" and "the" refer
to "one or more" when used in this application. Thus, for example,
reference to "a sample" includes a plurality of such samples, and
so forth.
[0044] As used herein, the term "about" can mean within 1 or more
standard deviation per the practice of the art. Alternatively,
"about" can mean a range of up to 20%. When particular values are
provided in the specification and claims the meaning of "about"
should be assumed to be within an acceptable error range for that
particular value.
[0045] The term "agent" refers to a molecule or a substance. An
agent as described herein "inhibits" the interaction between
IGFBP-3 and NONO. The term "inhibits" in this context thus refers
to slowing down or preventing the interaction. For example, an
agent that inhibits the interaction between IGFBP-3 and NONO as
described herein slows down or prevents IGFBP-3 binding to NONO,
and in this way diminishes or prevents DNA DSB repair mechanism,
preferably by NHEJ.
[0046] The term "chemosensitivity" and "radiosensitivity" as
referred to herein is the relative susceptibility of cells,
tissues, organs or organisms to the effect of chemotherapeutic
agents and ionizing radiation, respectively.
[0047] The term "peptide" as used herein includes but is not
limited to, two or more amino acids, or residues covalently linked
by a peptide bond or equivalent. In certain embodiments, amino
acids may be linked by non-natural and non-peptide bonds. In the
context of the present invention, it is to be understood that the
term "isolated" as used herein i.e. "an isolated peptide" is
intended to refer to a peptide that is separated from the natural
environment, e.g. the human body. The term "isolated peptide" as
used herein includes peptides based on the complete full-length
human IGFBP-3 sequence but are not part of the full protein, i.e.
they are isolated from it. In other words, isolated peptides
provided herein do not necessarily comprise the complete
full-length human IGFBP-3 sequence. However, the present invention
does not intend to exclude embodiments wherein the isolated peptide
is a portion of a larger peptide, such as a pre-pro-protein or a
polypeptide that comprises an amino acid sequence that can be
processed (e.g. by cleavage) into a number of smaller peptides
following expression. Isolated peptides described herein include
but are not limited to chemically synthesized peptides, recombinant
peptides, and peptides that have been modified. The person skilled
in the art will appreciate that a number of modifications can be
made to the peptides to improve peptide stability and
pharmacokinetic properties, for example, peptide absorption,
distribution, metabolism, and excretion (ADME) properties. The
peptides as herein described may be modified to form a cyclic
structure (i.e. a cyclic peptide). Methods of modifying a peptide
as herein disclosed to a cyclic structure are not particularly
limited and any suitable methods may be used. The peptides as
herein described may be modified such that the peptide includes
non-peptide bonds or other synthetic modifications such as the use
of non-natural amino acids. These modifications may render the
peptides more stable while in the body or more capable of
penetrating into cells.
[0048] The term "interaction" as used herein refers to either a
direct or indirect interaction. In the context of the present
invention, an agent that inhibits the "interaction" between IGFBP-3
and NONO therefore refers to, but is not limited to, either
inhibiting the physical binding of the two proteins (direct
interaction) or modulating the expression of one or both of the
proteins (indirect interaction). The term "interaction" as used
herein may also be taken to mean that the proteins exist as part of
the same multi-protein complex, independent of whether the proteins
are in direct physical contact. Protein interactions can be
determined by various methods including but is not limited to the
yeast two-hybrid system, affinity chromatography,
co-immunoprecipitation, proximity ligation assay, subcellular
fractionation and isolation of large molecular complexes. Each of
these methods is well characterised and can be readily performed by
one skilled in the art.
[0049] The term "conservative substitutions" used herein refers to
replacing one amino acid with another having similar structural
and/or chemical properties, such as the replacement of a leucine
with an isoleucine or valine, an aspartate with a glutamate, or a
threonine with a serine. A "conservative substitution" of a
particular sequence refers to substitution of those amino acids
that are not critical for peptide activity or substitution of amino
acids with other amino acids having similar properties, for example
acidic, basic, positively or negatively charged, polar or non-polar
etc, such that the substitution of even critical amino acids does
not reduce the activity of the peptide (i.e. the ability of the
peptide to inhibit NONO-IGFBP-3 interaction). Conservative
substitutions of functionally similar amino acids are well known in
the art. For example, the following six groups each contain amino
acids that are conservative substitutions for one another: i)
Alanine (A), Serine (S), Threonine (T); ii) Aspartic acid (D),
Glutamic acid (E); iii) Asparagine (N), Glutamine (Q); iv) Arginine
(R), Lysine (K); v) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); and vi) Phenylalanine (F), Tyrosine (Y), Tryptophan
(W). In some embodiments, individual substitutions of a single
amino acid or a small percentage of amino acids can also be
considered "conservative substitutions" if the substitution does
not reduce the activity of the peptide. The choice of conservative
amino acids may be selected based on the location of the amino acid
to be substituted in the peptide, for example if the amino acid is
on the exterior of the peptide and expose to solvents, or on the
interior and not exposed to solvents.
[0050] The three-letter abbreviations or one-letter abbreviations
of amino acids are known and standard in the art, and include for
example alanine (Ala or A); arginine (Arg or R); asparagine (Asn or
N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gln
or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His
or H); isoleucine (Ile or I): leucine (Leu or L); lysine (Lys or
K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro
or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or
W); tyrosine (Tyr or Y); and valine (Val or V). Non-traditional and
non-natural amino acids are also within the scope of the invention.
The amino acids described herein may be in the "L" or "D"
stereoisomeric form. In the absence of a "D" or "L" designation, an
amino acid in the three-letter abbreviation is in the "L" form.
[0051] Radiotherapy intended in the present invention is commonly
used in this technical field and can be performed according to
protocols known to those skilled in the art. For example,
radiotherapy as used herein includes but is not limited to
irradiation with cesium, iridium, iodine, cobalt or other suitable
isotopes. Radiotherapy may be systemic irradiation or local
irradiation. The dose fractionation and duration of the
radiotherapy intended in the present invention are not particularly
limiting. Exemplary methods include radiotherapy divided into 25 to
30 fractions, over about 5 to 6 weeks, and performed for 2 to 3
minutes per day.
[0052] As used herein, the term "radiomimetic agent" refers to
cytotoxic agents that damage DNA in such a way that the lesions
produced in DNA are similar to those resulting from ionising
radiation. Examples of radiomimetic agents which cause DNA strand
breaks include but is not limited to bleomycin, doxorubicin
(adriamycin), 5-fluorouracil (5 FU), neocarzinostatin, alkylating
agents and other agents that produce DNA adducts.
[0053] As used herein, the term "chemotherapeutic agent" includes
but is not limited to a compound that introduces DNA double strand
breaks, for example, bifunctional alkylator, a topoisomerase
inhibitor, a monofunctional alkylator, an antimetabolite, a
replication inhibitor and a platinum drug. The chemotherapeutic
agent as used herein may be temozolomide, etoposide, doxorubicin,
gemcitabine, cisplatin or carboplatin.
[0054] The term "PARP" as used herein refers to the enzyme family
of poly (ADP-ribose) polymerases (PARP). Enzymes of the PARP family
include but are not limited to PARP1, PARP2 and PARP3. PARP
inhibitors which may be used in accordance with the invention
include but are not limited to veliparib, olaparib and
talazoparib.
[0055] Methods of generating the peptides as described herein are
not particularly limiting. Exemplary methods include solid phase
peptide synthesis and solution phase peptide synthesis.
[0056] Also contemplated are pharmaceutically acceptable salts of
the peptide provided herein. The term "pharmaceutically acceptable
salt" includes both acid and base addition salts and refers to
salts which retain the biological effectiveness and properties of
the free bases or acids, and which are not biologically or
otherwise undesirable. The pharmaceutically acceptable salts are
formed with inorganic or organic acids or bases and can be prepared
in situ during the final isolation and purification of the
compounds, or by separately reacting a purified compound in its
free base or acid form with a suitable organic or inorganic acid or
base, and isolating the salt thus formed.
[0057] As used herein "pharmaceutical composition" or "composition"
refers to a mixture of at least one agent as described herein, one
peptide as described herein, or pharmaceutically acceptable salts,
solvates, hydrates thereof, with other chemical components, such as
pharmaceutically acceptable excipients. Pharmaceutical compositions
suitable for the delivery of the agents and peptides as described
herein and methods for their preparation will be apparent to those
skilled in the art.
[0058] Also contemplated are pharmaceutical compositions comprising
at least an agent or a peptide provided herein, further comprising
one or more chemotherapeutic agent, radiomimetic agent and/or a
PARP inhibitor and optionally at least one pharmaceutical
excipient. The term "pharmaceutically acceptable excipient" refers
to any pharmaceutically acceptable inactive component of the
composition. As is known in the art, excipients include diluents,
buffers, binders, lubricants, disintegrants, colorants,
antioxidants/preservatives, pH-adjusters, etc. The excipients are
selected based on the desired physical aspects of the final form:
e.g. a parenteral formulation for injection, obtaining a tablet
with desired hardness and friability being rapidly dispersible and
easily swallowed, and the like. Suitable forms of a pharmaceutical
composition may include, but is not limited to, a tablet, capsule,
elixir, liquid formulation, delayed or sustained release, and the
like. The physical form and/or content of a pharmaceutical
composition contemplated are conventional preparations that may be
formulated by those skilled in the pharmaceutical formulation
field.
[0059] A cancer described herein as expressing IGFBP-3, includes a
cancer cell population that is tumorigenic, including benign
tumours and malignant tumours, or non-tumorigenic, in which at
least 5% of the observed cells have the capability of producing the
IGFBP-3 protein. Methods of determining IGFBP-3 expression in
cancer are not particularly limiting. Exemplary methods include
western blotting, immunohistochemistry or immunocytochemistry and
PCR (polymerase chain reaction). Exemplary cancers include but are
not limited to breast cancer, triple negative breast cancer (TNBC),
prostate cancer, pancreatic cancer or glioblastoma cancer.
[0060] It is also contemplated that an agent or a peptide provided
herein may be delivered to a cancer cell in-vitro or in-vivo. In
some embodiments, an agent or a peptide provided herein is
administered to an IGFBP-3 expressing cancer cell in-vitro or
in-vivo. In certain embodiments, an agent or a peptide provided
herein is administered to an IGFBP-3 expressing cancer cell
in-vitro or in-vivo and inhibits the NONO-IGFBP-3 interaction in
the cancer cell thereof. An agent or peptide provided herein may be
administered to a cell with a pharmaceutically acceptable carrier
within a composition as herein described.
[0061] A "subject" to be treated by a method described herein
includes mammal, including a human ("patient") or non-human subject
(for example, cat, dog, and the like). An agent, peptide or
composition herein described may be administered to a human or
non-human subject. An agent, peptide or composition herein
described may be administered to a human cancer cell or a non-human
cancer cell in vitro or in vivo. In some embodiments, the cell is a
mammalian cell.
[0062] As used herein, a "therapeutically effective amount" of an
agent, peptide or composition herein described includes an amount,
when administered (whether as a single dose or as a time course of
multiple treatments), prevents disease advancement or promotes
disease regression evidenced by a decrease in severity of disease
symptoms, an increase in frequency and duration of disease
symptom-free periods, or a prevention of impairment or disability
due to the disease affliction. A therapeutically effective amount
of an agent, peptide or composition herein described includes a
"prophylactically effective amount" which is any amount of an
agent, peptide or composition described herein that, when
administered to a subject at risk of developing a disease or of
suffering a recurrence of disease, inhibits the development or
recurrence of the disease. The ability of a therapeutic agent to
promote disease regression or inhibit the development or recurrence
of the disease may be evaluated using a variety of methods known to
the skilled practitioner, such as animal model systems predictive
of efficacy in humans, by assaying the activity of the agent in
in-vitro assays, or the like. By way of example for the treatment
of cancer, a therapeutically effective amount of an agent, peptide
or composition as described herein may enhance chemosensitivity or
radiosensitivity such that cancer cell growth is reduced by at
least about 20%, by at least about 40%, by at least about 60%, or
by at least about 80% relative to untreated cancer cells.
Alternatively, a therapeutically effective amount of an agent,
peptide or composition as herein described may, when used in
conjunction with radiotherapy, chemotherapy and/or a PARP
inhibitor, allow the dose and/or duration of the radiotherapy,
chemotherapy or PARP inhibitor treatment to be decreased while
still achieving the same clinical benefit. A therapeutically
effective amount of an agent, peptide or composition herein
described may enhance chemosensitivity or radiosensitivity such
that cancer cell growth is inhibited or reduced to a statistically
significant degree of cell growth or tumour growth as compared to
control. "Statistical significance" means significance at the p
<0.05 level, or such other measure of statistical significance
as would be used by those of skill in the art of biomedical
statistics in the context of a particular type of treatment or
prophylaxis.
[0063] Depending upon the cancer type as described herein, the
route of administration and/or whether the agent, peptide or
composition as herein described is administered locally or
systemically, a wide range of permissible dosages are contemplated.
The dosages may be single or divided and may be administered
according to a wide variety of protocols, including q.d. (once a
day), b.i.d. (two times a day), t.i.d. (three times a day), or even
every other day, biweekly (b.i.w.), once a week, once a month, once
a quarter, and the like. In each of these cases, it is understood
that the therapeutically effective amounts described herein
correspond to the instance of administration, or alternatively to
the total daily, weekly, month, or quarterly dose, as determined by
the dosing protocol.
[0064] It is contemplated that an agent, peptide or composition as
herein described may be administered with one or more
chemotherapeutic agent, radiotherapy and/or a PARP inhibitor.
Administration as an agent, peptide or composition as herein
described with one or more chemotherapeutic agent or PARP inhibitor
include but is not limited to simultaneous administration, separate
administration or sequential administration. The term
"simultaneously" in the context of drug administration refers to an
administration of at least 2 active ingredients by the same route
and at the same time or at substantially the same time. The term
"separately" in the context of drug administration refers to an
administration of at least 2 active ingredients at the same time or
at substantially the same time by different routes. The term
"sequentially" in the context of drug administration refers to an
administration of at least 2 active ingredients at different times,
the administration route being identical or different. An agent,
peptide or composition as herein described can be administered
simultaneously with radiotherapy, either before or after
radiotherapy.
[0065] An agent or composition thereof as described herein may be
administered for example orally, intravenously, intramuscularly,
intraperitoneally or subcutaneously. A peptide or composition
thereof as described herein may be administered intravenously.
BRIEF DESCRIPTION OF THE FIGURES
[0066] Embodiments of the invention will now be described, by way
of example only, with reference to the accompanying drawings as
follows.
[0067] FIG. 1a to 1f: IGFBP-3 forms a complex with NONO in response
to etoposide treatment. (a) MDA-MB-468 basal-like TNBC cells were
exposed to 20 .mu.M etoposide (Etop) for the indicated times, and
NONO was precipitated from cell lysates by anti-IGFBP-3 antiserum
(Fab fraction) coupled to agarose beads. Uncoupled agarose beads
were used for immunoprecipitation controls. Samples were blotted
for NONO after fractionation by SDS-PAGE. Panels on right show
blots of whole-cell lysates without immunoprecipitation. Molecular
weight markers are shown on the left. (b) IGFBP-3 was downregulated
in MDA-MB-468 cells by siRNA, and cell lysates immunoprecipitated
with IGFBP-3-Fab beads 2 h after etoposide stimulation.
Precipitates were blotted for NONO and IGFBP-3. (c) MDA-MB-468
cells were treated with etoposide, and nuclear extracts were
prepared and immunoprecipitated with IGFBP-3-Fab beads. Panels on
left show NONO, lamin B1 (nuclear marker), and GAPDH (cytoplasmic
marker) in whole nuclear extracts (5% of immunoprecipitated
sample). GAPDH in the whole cytoplasmic fraction, run on the same
gel, is also shown for comparison. Panels on right show the same
proteins after IP. 0 and 4 h time points are shown from a 4-h
time-course. For each analyte, all samples (input and IP) were run
on the same gel. For NONO, but not lamin B1 or GAPDH, the input
blots shown were from shorter exposures, to avoid saturating the
images. (d) Similar experiment to that shown in FIG. 1a, but in
HCC1806 basal like TNBC cells. (e) Quantitation of bands
immunoblotted for NONO in HCC1806 cells. Data are mean band
density.+-.SEM from 5 experiments. *P<0.05 vs. time 0 by post
hoc Fisher's LSD test after ANOVA. (f) Binding of recombinant
IGFBP-3 to immobilized recombinant NONO, measured in an ELISA
format in which bound IGFBP-3 is immunodetected and quantitated
colorimetrically at 450 nm. Panels show dose--response curves for
NONO (left) and IGFBP-3 (right).
[0068] FIGS. 2a and 2b: IGFBP-3 complexes with NONO visualized by
proximity ligation assay. (a) MDA-MB-468 and (b) HCC1806 TNBC cells
were exposed to 20 .mu.M etoposide (Etop) for the indicated times,
and bimolecular interactions, shown as red dots, between IGFBP-3
and NONO, as indicated, were measured by PLA. Bar 25 .mu.m.
[0069] FIGS. 3a to 3e: PARP inhibition blocks IGFBP-3 complex
formation with NONO. (a) MDA-MB-468 cells were incubated for 24 h
with 20 .mu.M veliparib (ABT-888) before exposure to 20 .mu.M
etoposide. NONO was immunoprecipitated with IGFBP-3-Fab beads and
detected by immunoblotting. (b) Quantitation of bands immunoblotted
for NONO in MDA-MB-468 cells. Data are mean band density.+-.SEM
from 4 experiments. *P<0.05, **P<0.005 vs. the corresponding
time 0 by post hoc Fisher's LSD test after ANOVA. NS, not
significant. (c) The PARP inhibitor olaparib also inhibits
NONO-IGFBP-3 interaction. MDA-MB-468 cells were incubated 24 h with
10 .mu.M olaparib before exposure to 20 .mu.M etoposide. (d) PLA
showing interactions (yellow dots) between IGFBP-3 and NONO in
MDA-MB-468 cells (above) and HCC1806 cells (below) after 2 h
treatment with 20 .mu.M etoposide, following 24 h
preincubation.+-.20 .mu.M veliparib. Blue=nuclei (DAPI). Bar 20
.mu.m. Confocal images superimposed over phase contrast images. (e)
Quantitation of inhibition by 20 .mu.M veliparib of IGFBP-3
interaction with NONO over 4 h of etoposide treatment in MDA-MB-468
cells, measured by PLA; 5 fields (.about.20 nuclei/field) counted
for each condition and each timepoint in each experiment. Data are
mean values.+-.SEM from 3 experiments. *P<0.001 vs. the
corresponding time 0 by post hoc Fisher's LSD test after ANOVA. NS,
not significant.
[0070] FIGS. 4a to 4f: PARP inhibition decreases DNA end-joining in
TNBC cells. (a) Upper panels: .gamma.H2AX immunofluorescence in
MDA-MB-468 cells at time 0 (i.e. 1 h after exposure to etoposide)
and after 4 h of recovery (T4). Cells were pre-treated with
olaparib (10 .mu.M) or veliparib (20 .mu.M) as indicated. Bar 20
.mu.m. Lower panels: representative images at higher power of T0
cells.+-.etoposide, to illustrate the punctate .gamma.H2AX
fluorescence. Bar 10 .mu.m. (b) Mean fluorescence values (arbitrary
units).+-.SEM are shown from 3 experiments. (c) and (d) .gamma.H2AX
immunofluorescence in HCC1806 cells, with quantitation from 3
experiments, details are as described for panels (a) and (b). Bar
20 .mu.m. ANOVA with Fisher's LSD post hoc LSD test: *(blue)
P<0.05 vs. T0+etoposide; *(red) P<0.05 vs. T4 +etoposide;
*P<0.05 vs. the corresponding T0 value. (e) DNA end-joining
assay: cells were treated with inhibitors (20 .mu.M veliparib or 10
.mu.M olaparib) or no inhibitor (Con) for 24 h, then exposed to 20
.mu.M etoposide for 2 h. In control lanes (right), DNA or nuclear
extract (NE) has been omitted. After adding nuclear extract for 30
min, substrate DNA was added and end joining proceeded for 30 min
at 25.degree. C. A representative gel is shown for MDA-MB-468
cells. Black arrows show the bands quantitated. Open arrow show
size markers in kb. All lanes are from a single gel. (f) Upper
panel: Quantitation of end-joining activity 2 h after etoposide in
MDA-MB-468 cells, mean.+-.SEM, n=3. Lower panel: Quantitation of
end-joining activity 2 h after etoposide in HCC1806 cells,
mean.+-.SEM, n=5. *P<0.05 vs. control by post hoc Fisher's LSD
test after ANOVA.
[0071] FIGS. 5a to 5f: The effects of inhibitory peptides on
NONO-IGFBP-3 interaction. (a) NONO-IGFBP-3 binding assay was used
to screen inhibitory peptides. The inhibitory effect of peptides
#64 to #67 towards the binding of IGFBP-3 with NONO was
investigated and compared to no peptide added. The absorbance
values are means of duplicate runs in a single assay. (b) Peptide
#66 showed a consistent inhibitory effect suggested by
significantly reduced absorbance value (P=0.014), indicative of
reduced binding between IGFBP-3 and NONO (n=3). (c) and (d)
.gamma.H2AX signal was measured in 2 BRCA wild-type breast cancer
cell lines, MDA-MB-468 and HCC1806, respectively, exposed to 25
.mu.M etoposide, after 16 h incubation with the indicated peptides
at 10 .mu.M, or no peptide. Peptides #65 and #66 with overlapping
residues HLKFLNVLS (His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser) caused a
sustained .gamma.H2AX signal compared to no peptide or peptide #64
in which a low .gamma.H2AX signal was observed. An elevated
.gamma.H2AX signal is indicative of sustained DNA damage, i.e.
inhibition of DNA repair, compared to the control cells. Peptide
#66 was further evaluated in the presence of PARP inhibitors, (e)
veliparib (ABT-888) and (f) olaparib (Lynparza). The .gamma.H2AX
signal in the absence of peptide #66 is expressed as 100%, and bars
represent the ratio of .gamma.H2AX signal in the presence of the
peptide, to that in its absence. Preincubation with either PARP
inhibitor in the presence of peptide #66 increased the .gamma.H2AX
signal by almost 2-fold at 10 .mu.M of PARP inhibitor, suggesting a
combination effect between the PARP inhibitors and the inhibitor of
NONO-IGFBP-3 interaction i.e. peptide #66.
[0072] FIGS. 6a and 6b: Peptide #66 inhibits the formation of DNA
repair complexes in TNBC cell lines, MDA-MB-468 and HCC1806. (a) In
MDA-MB-468 breast cancer cells, exposure to 20 .mu.M of etoposide
increased the interaction between IGFBP-3 and NONO/SFPQ, which
peaked 2 h after etoposide treatment (black arrow). Treatment with
10 .mu.M of peptide #66 for 1 h prior to etoposide exposure
decreased the formation of the IGFBP-3--NONO/SFPQ complex (white
arrow). (b) In HCC1806 breast cancer cells, exposure to 20 .mu.M
etoposide increased the interaction between IGFBP-3 and NONO/SFPQ,
which peaked 2 h after etoposide treatment (black arrow). Treatment
with 20 .mu.M of peptide #66 for 1 h prior to etoposide exposure
decreased the formation of the IGFBP-3--NONO/SFPQ complex (white
arrow).
[0073] FIGS. 7a and 7b: Peptide #66 makes breast cancer cells more
responsive to the effect of a PARP inhibitor (PARPi) on cell
survival following chemotherapy treatment. (a) In HCC1806 breast
cancer cells, in the absence of a PARPi, peptide #66 (20 .mu.M) had
no effect on cell survival over 14 days (colony number) either
before or after etoposide treatment (Etop; 100 nM). In cells
treated with a PARPi (veliparib, 5 peptide #66 similarly had no
effect on cell survival without etoposide treatment, but after
etoposide treatment, peptide #66 significantly decreased cell
survival over the effect of the PARPi alone. This indicates that,
in the presence of PARPi, peptide #66 can increase cell
responsiveness to chemotherapy (etoposide). (a) In the bar graph
starting from the left, the 1.sup.st, 3.sup.rd, 5.sup.th, and
7.sup.th bars represent conditions without peptide #66 and the
2.sup.nd, 4.sup.th, 6.sup.th and 8.sup.th bars represent conditions
in the presence of peptide #66. Mean data.+-.SEM from 4 assays,
each in triplicate, P<0.001. (b) Representative images of cell
colonies after 14 days.
[0074] FIGS. 8a and 8b: Peptide #66 diffuses rapidly into the
nuclei of breast cancer cells. (a) Fixed cell imaging: peptide #66
(5 .mu.M) labelled biosynthetically with 5-TAMRA (provided by Dr Yu
Heng Lau, School of Chemistry, University of Sydney) was added to
cultures of HCC1806 breast cancer cells for the indicated times,
after which cells were fixed and imaged by confocal microscopy.
Images show cell nuclei (blue) and labelled peptide #66 (green);
colocalized peptide and nuclei appear cyan, and peaked at 30 min.
(b) Live cell imaging: peptide #66 (2.5 .mu.M) labelled
biosynthetically with 5-TAIVIRA was added to cultures of HCC1806
breast cancer cells for the indicated times, after which cells were
imaged by confocal microscopy without fixation. Images show
increasing colocalization of labelled peptide #66 (red) with cell
nuclei (blue).
[0075] FIGS. 9a and 9b: Peptide #66 inhibits the formation of DNA
repair complexes in glioblastoma cell lines, A172 and M059K. (a) In
2 human glioblastoma cell lines, A172 and M059K, treatment with 20
.mu.M of etoposide induced a complex between IGFBP-3 and NONO/SFPQ,
determined by coimmunoprecipitation. Complex formation appeared
maximal at 2 h in A172 and 1 h in M059K (black arrows). (b) In
M059K glioblastoma cells, formation of the complex between IGFBP-3
and NONO/SFPQ peaked at 1 h (black arrow) and was inhibited by
incubation with 25 .mu.M of peptide #66 (white arrow).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0076] In one embodiment, provided is an agent that inhibits the
interaction between IGFBP-3 and NONO. In some embodiments, the
agent is a small molecule. In certain embodiments, the agent is a
substance or a compound that inhibits the interaction between
IGFBP-3 and NONO.
[0077] In a further embodiment, provided is an isolated peptide
comprising residues:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9,
[0078] wherein X.sub.1 is His, X.sub.2 is Leu, X.sub.3 is Lys,
X.sub.4 is Phe, X.sub.5 is Leu, X.sub.6 is Asn, X.sub.7 is Val,
X.sub.8 is Leu and X.sub.9 is Ser, or conservative substitutions
thereof,
[0079] or a pharmaceutically acceptable salt of the peptide.
[0080] In some embodiments, provided is a peptide comprising the
sequence:
His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser-Pro-Arg-Gly,
[0081] or conservative substitutions thereof,
[0082] or a pharmaceutically acceptable salt of the peptide.
[0083] In some embodiments, provided is a peptide comprising the
sequence:
Thr-Leu-Asn-His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser,
[0084] or conservative substitutions thereof,
[0085] or a pharmaceutically acceptable salt of the peptide.
[0086] In one embodiment, provided is an isolated peptide
comprising residues:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9,
[0087] wherein X.sub.1 is His, X.sub.2 is Leu, X.sub.3 is Lys,
X.sub.4 is Phe, X.sub.5 is Leu, X.sub.6 is Asn, X.sub.7 is Val,
X.sub.8 is Leu and X.sub.9 is Ser, or conservative substitutions
thereof, [0088] or a pharmaceutically acceptable salt of the
peptide, wherein the peptide inhibits the interaction between
IGFBP-3 and NONO.
[0089] In some embodiments, provided is an isolated peptide
comprising the sequence His-Leu, Leu-Lys, Lys-Phe, Phe-Leu,
Leu-Asn, Asn-Val, Val-Leu or Leu-Ser, or conservative substitutions
thereof, or a pharmaceutically acceptable salt of the peptide,
wherein the peptide inhibits the interaction between IGFBP-3 and
NONO.
[0090] In some embodiments, provided is an isolated peptide
comprising the sequence His-Leu-Lys, Leu-Lys-Phe, Lys-Phe-Leu,
Phe-Leu-Asn, Leu-Asn-Val, Asn-Val-Leu, or Val-Leu-Ser, or
conservative substitutions thereof, or a pharmaceutically
acceptable salt of the peptide, wherein the peptide inhibits the
interaction between IGFBP-3 and NONO.
[0091] In some embodiments, provided is an isolated peptide
comprising the sequence His-Leu-Lys-Phe, Leu-Lys-Phe-Leu,
Lys-Phe-Leu-Asn, Phe-Leu-Asn-Val, Leu-Asn-Val-Leu, or
Asn-Val-Leu-Ser, or conservative substitutions thereof, or a
pharmaceutically acceptable salt of the peptide, wherein the
peptide inhibits the interaction between IGFBP-3 and NONO.
[0092] In some embodiments, provided is an isolated peptide
comprising the sequence His-Leu-Lys-Phe-Leu, Leu-Lys-Phe-Leu-Asn,
Lys-Phe-Leu-Asn-Val, Phe-Leu-Asn-Val-Leu, or Leu-Asn-Val-Leu-Ser,
or conservative substitutions thereof, or a pharmaceutically
acceptable salt of the peptide, wherein the peptide inhibits the
interaction between IGFBP-3 and NONO.
[0093] In some embodiments, provided is an isolated peptide
comprising the sequence His-Leu-Lys-Phe-Leu-Asn,
Leu-Lys-Phe-Leu-Asn-Val, Lys-Phe-Leu-Asn-Val-Leu, or
Phe-Leu-Asn-Val-Leu-Ser, or conservative substitutions thereof, or
a pharmaceutically acceptable salt of the peptide, wherein the
peptide inhibits the interaction between IGFBP-3 and NONO.
[0094] In some embodiments, provided is an isolated peptide
comprising the sequence His-Leu-Lys-Phe-Leu-Asn-Val,
Leu-Lys-Phe-Leu-Asn-Val-Leu, or Lys-Phe-Leu-Asn-Val-Leu-Ser, or
conservative substitutions thereof, or a pharmaceutically
acceptable salt of the peptide, wherein the peptide inhibits the
interaction between IGFBP-3 and NONO.
[0095] In some embodiments, provided is an isolated peptide
comprising the sequence His-Leu-Lys-Phe-Leu-Asn-Val-Leu, or
Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser, or conservative substitutions
thereof, or a pharmaceutically acceptable salt of the peptide,
wherein the peptide inhibits the interaction between IGFBP-3 and
NONO.
[0096] In some embodiments, provided is an isolated peptide
comprising any one or more of the following residues:
X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6, X.sub.7,
X.sub.8, X.sub.9,
[0097] wherein X.sub.1 is His, X.sub.2 is Leu, X.sub.3 is Lys,
X.sub.4 is Phe, X.sub.5 is Leu, X.sub.6 is Asn, X.sub.7 is Val,
X.sub.8 is Leu and X.sub.9 is Ser, or conservative substitutions
thereof,
[0098] or a pharmaceutically acceptable salt of the peptide,
wherein the peptide inhibits the interaction between IGFBP-3 and
NONO.
[0099] In some embodiments, provided is a peptide comprising the
sequence:
His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser-Pro-Arg-Gly,
[0100] or conservative substitutions thereof, [0101] or a
pharmaceutically acceptable salt of the peptide, wherein the
peptide inhibits the interaction between IGFBP-3 and NONO.
[0102] In some embodiments, provided is a peptide comprising the
sequence:
Thr-Leu-Asn-His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser,
[0103] or conservative substitutions thereof, [0104] or a
pharmaceutically acceptable salt of the peptide, wherein the
peptide inhibits the interaction between IGFBP-3 and NONO.
[0105] In some embodiments, the peptide of the present disclosure
is about 5-50 amino acids in length, such as 5-45 amino acids, 5-40
amino acids, 5-35 amino acids, 5-30 amino acids, 5-25 amino acids,
5-20 amino acids, 5-15 amino acids, or 5-10 amino acids.
Preferably, the peptide of the present disclosure is about 9 amino
acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino
acids, 14 amino acids, or 15 amino acids in length. More
preferably, the peptide of the present disclosure is about 12 amino
acids in length.
[0106] In certain embodiments, the peptide of the present
disclosure comprises the amino acid sequence
Thr-Leu-Asn-His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser or a sequence
having at least about 80% identity, such as at least about 85%
identity, at least about 90% identity or at least about 95%
identity to the amino acid sequence
Thr-Leu-Asn-His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser. In some
embodiments, the peptide of the present disclosure comprises the
amino acid sequence His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser-Pro-Arg-Gly
or a sequence having at least about 80% identity, such as at least
about 85% identity, at least about 90% identity or at least about
95% identity to the amino acid sequence
His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser-Pro-Arg-Gly. In some
embodiments, the peptide of the present disclosure comprises the
amino acid sequence
Thr-Leu-Asn-His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser-Pro-Arg-Gly or a
sequence having at least about 80% identity, such as at least about
85% identity, at least about 90% identity or at least about 95%
identity to the amino acid sequence
Thr-Leu-Asn-His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser-Pro-Arg-Gly. In
some embodiments, the peptide of the present disclosure comprises
the amino acid sequence His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser or a
sequence having at least about 80% identity, such as at least about
85% identity, at least about 90% identity or at least about 95%
identity to the amino acid sequence
His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser.
[0107] In one embodiment, provided is a pharmaceutical composition
comprising an agent of the invention, or an isolated peptide of the
invention and optionally at least one pharmaceutically acceptable
excipient. In some embodiments, the pharmaceutical composition
further comprises a chemotherapeutic agent, a radiomimetic agent or
a PARP inhibitor. In a related embodiment, the chemotherapeutic
agent is selected from the group consisting of a bifunctional
alkylator, a monofunctional alkylator, a topoisomerase inhibitor,
an antimetabolite, a replication inhibitor and a platinum drug. In
some embodiments, the chemotherapeutic agent is etoposide. In
certain embodiments, the PARP inhibitor is veliparib. In some
embodiments, the PARP inhibitor is olaparib. In some embodiments,
the PARP inhibitor is talazoparib.
[0108] In a further embodiment, provided is a method of enhancing
chemosensitivity or radiosensitivity in cancer treatment comprising
administering to a subject in need thereof a therapeutically
effective amount of an agent of the invention, an isolated peptide
of the invention or a pharmaceutical composition of the invention,
wherein the cancer is an IGFBP-3 expressing cancer. In some
embodiments, the IGFBP-3 expressing cancer is breast cancer,
prostate cancer, pancreatic cancer or glioblastoma cancer. In
certain embodiments, the IGFBP-3 expressing cancer is Triple
Negative Breast Cancer (TNBC).
[0109] In one embodiment, provided is a method of enhancing
chemosensitivity or radiosensitivity in TNBC treatment comprising
administering to a subject in need thereof a therapeutically
effective amount of an agent of the invention, an isolated peptide
of the invention or a pharmaceutical composition of the
invention.
[0110] In one embodiment, provided is a method of treating cancer
comprising administering to a subject in need thereof a
chemotherapeutic agent and an agent of the present disclosure or a
peptide of the present disclosure. In some embodiments, the cancer
of the present disclosure may be mediated by IGFBP-3 and/or
NONO/SFPQ. In a related embodiment, the cancer is an IGFBP-3
expressing cancer.
[0111] In one embodiment, provided is a method of treating cancer
comprising administering to a subject in need thereof radiotherapy
and an agent of the present disclosure or a peptide of the present
disclosure. In some embodiments, the cancer of the present
disclosure may be mediated by IGFBP-3 and/or NONO/SFPQ. In a
related embodiment, the cancer is an IGFBP-3 expressing cancer.
[0112] In one embodiment, provided is a method of treating cancer
comprising administering to a subject in need thereof a
radiomimetic agent and an agent of the present disclosure or a
peptide of the present disclosure. In some embodiments, the cancer
of the present disclosure may be mediated by IGFBP-3 and/or
NONO/SFPQ. In a related embodiment, the cancer is an IGFBP-3
expressing cancer.
[0113] In one embodiment, provided is a method of inhibiting an
interaction between IGFBP-3 and NONO in a cell comprising
administering to the cell an agent of the present disclosure or a
peptide of the present disclosure. Preferably, the cell is a human
cell. More preferably, the cell is in a human body.
[0114] In one embodiment, provided is a method of preventing or
suppressing DNA DSB repair in a cell comprising administering to
the cell an agent of the present disclosure or a peptide of the
present disclosure. Preferably, the cell is a human cell. More
preferably, the cell is in a human body.
[0115] In one embodiment, provided is use of an agent of the
invention, or an isolated peptide of the invention in the
manufacture of a medicament for enhancing chemosensitivity or
radiosensitivity in cancer treatment, wherein the cancer is an
IGFBP-3 expressing cancer.
[0116] In one embodiment, provided is use of an agent of the
invention, or an isolated peptide of the invention in the
manufacture of a medicament for enhancing chemosensitivity or
radiosensitivity in TNBC treatment.
[0117] In some embodiments, provided is an agent of the invention
for use in a method of enhancing chemosensitivity or
radiosensitivity in cancer treatment, wherein the cancer is an
IGFBP-3 expressing cancer. In a related embodiment, the IGFBP-3
expressing cancer is breast cancer, prostate cancer, pancreatic
cancer or glioblastoma cancer.
[0118] In some embodiments, provided is an agent of the invention
for use in a method of enhancing chemosensitivity or
radiosensitivity in TNBC treatment.
[0119] In certain embodiments, provided is an isolated peptide of
the invention or a pharmaceutically acceptable salt thereof for use
in a method of enhancing chemosensitivity or radiosensitivity in
cancer treatment, wherein the cancer is an IGFBP-3 expressing
cancer. In a related embodiment, the IGFBP-3 expressing cancer is
breast cancer, prostate cancer, pancreatic cancer or glioblastoma
cancer.
[0120] In some embodiments, provided is an isolated peptide of the
invention or a pharmaceutically acceptable salt thereof for use in
a method of enhancing chemosensitivity or radiosensitivity in TNBC
treatment.
[0121] Further preferred embodiments of the invention will now be
described, by way of example only, with reference to the
accompanying drawings.
EXAMPLES
[0122] General Material
[0123] Etoposide was obtained from Sigma-Aldrich (St. Louis, Mo.,
USA). Veliparib (ABT-888) was from Selleckchem, Houston, Tex., USA
and olaparib from AdooQ Bioscience, Irvine, Calif. Rabbit antiserum
R-100 against full-length human IGFBP-3, and recombinant human
IGFBP-3 expressed in human cells, were prepared in-house.
Recombinant human NONO, Myc-DDK tagged (TP326567) was obtained from
Origene, Rockville, Md., USA. FLAG antibody plates (L00455C) were
from GenScript, Piscataway, N.J., USA. Goat anti-rabbit IgG-HRP
(ab97080) was from Abcam, Melbourne, VIC, Australia, and 1-Step
Turbo TMB-ELISA substrate solution was from ThermoFisher, Scoresby,
VIC, Australia.
[0124] Cell Culture
[0125] The human basal-like triple negative breast cancer (TNBC)
cell lines MDA-MB-468 and HCC1806 were obtained from ATCC,
Manassas, Va. and maintained in RPMI 1640 medium containing 5% FBS
and 10 .mu.g/mL bovine insulin under standard conditions.
Cryopreserved stocks were established within 1 month of receipt,
and fresh cultures for use in experiments were established from
these stocks every 2 to 3 months. All cell lines tested negative
for mycoplasma. Inhibitor treatments were carried out for 24 h with
veliparib (20 .mu.M), olaparib (10 .mu.M), followed by etoposide
(20 .mu.M).
[0126] siRNA Mediated Transient Knockdown
[0127] IGFBP-3 was downregulated using siRNAs from Qiagen (Hilden,
Germany) (Table 1). Transfection was performed by electroporation
(Amaxa Nucleofector, Lonza, Cologne, Germany). In brief, the cells
were harvested by trypsinization and resuspended at
1.times.10.sup.6 cells in 100 .mu.L Transfection Reagent solution V
(Lonza) and mixed with 100 Nm targeting siRNA or AllStars negative
control siRNA (Qiagen). Immediately after electroporation, cells
were transferred to complete medium and plated for analysis.
Knockdown was confirmed by qRT-PCR as previously described (Martin
J L et al., Mol Cancer Therap., 2014, 13, 316-328) using Taqman
probe Hs00181211_m1 for IGFBP-3 and hydroxymethylbilane synthase
(HMBS; Hs00609297_m1) as an internal control (Applied Biosystems,
Foster City, Calif., USA).
TABLE-US-00001 TABLE 1 IGFBP-3 siRNAs IGFBP-3 Sense strand siRNA
#1.sup.a 5'GUU GAC UAC GAG UCU CAG AUU 3' siRNA #2.sup.b 5'AGG UUA
AUG UGG AGC UCA AUU 3' .sup.aDesigned by Qiagen (Hilden, Germany)
.sup.bCatalog No. SI02780589, Qiagen
[0128] Co-Immunoprecipitation and Western Blotting
[0129] Immunoprecipitation of IGFBP-3 complexes using anti-IGFBP-3
IgG (Fab fraction) coupled to agarose beads was performed as
previously described (Lin M Z et al., Oncogene, 2014, 33, 85-96).
For immunoprecipitations using NONO, cells
(.about.1.times.10.sup.6) were lysed in 1 mL ice-cold RIPA lysis
buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, mM EDTA, 1% Triton
X-100) supplemented with protease (cOmplete.TM. Mini) and
phosphatase (PhosSTOP.TM.) inhibitors (Roche; Sigma-Aldrich,
Sydney, Australia) at 4.degree. C. for 1 h and spun at
10,000.times.g for 10 min to pellet cell debris. Lysates were
precleared by mixing with 20 .mu.L of Protein A agarose beads
(Roche; Sigma-Aldrich) for 1 h at 4.degree. C. Pre-cleared lysates
were mixed overnight with specific antibodies and Protein A agarose
beads (blocked by mixing with 1% BSA in RIPA buffer for 1 h at
4.degree. C.). The antibody used for IP was NONO [N-terminal]
(Sigma-Aldrich #N8789), 2.5 .mu.g per sample. To prepare nuclear
extracts for coIP, cellular fractionation was performed according
to the manufacturer's protocol for the NE-PER Nuclear and
Cytoplasmic Extraction Kit (ThermoFisher). Immunoprecipitated
samples were resuspended in Laemmli sample buffer containing 50 mM
dithiothreitol, heated at 95-100.degree. C. for 6 min, and
fractionated on 12% SDS-PAGE gels. Proteins were transferred to
Protran.RTM. supported nitrocellulose membranes (Amersham, UK) at
160 mA for 2 h. Membranes were blocked in 50 g/L skim milk powder
and probed with primary antibodies (NONO (as above), 1:2000;
IGFBP-3 [C19], 1:750, Santa Cruz Biotechnology #sc-6003; GAPDH
[14C10], 1:2000, Cell Signaling #2118; and Lamin B1, 1:2000, Abcam
#ab16048) at 4.degree. C. for 16 h. Immunoreactive bands were
visualized as previously described (Lin M Z et al., Oncogene, 2014,
33, 85-96).
[0130] Proximity Ligation Assay (PLA)
[0131] PLA was performed using the Duolink Detection Kit (Olink
Bioscience Uppsala, Sweden) as previously described (Lin M Z et
al., Oncogene, 2014, 33, 85-96). Briefly, cells were grown on 8-mm
glass coverslips to 50% confluency, treated, and prepared for
microscopy by fixing, permeabilizing and blocking. Coverslips were
incubated with primary antibody pairs (raised in different species)
targeting the proteins under investigation overnight at 4.degree.
C. 1:500; NONO (as above) and 1:500; IGFBP-3 (as above), 1:100.
This was followed by incubation with PLA probes MINUS and PLUS for
1 h at 37.degree. C., probe ligation for 30 min at 37.degree. C.
and amplification over 100 min at 37.degree. C. Interactions were
detected as amplified far-red signals using a Leica TCS SP5
confocal microscope (Leica Microsystems, Wetzlar, Germany) and
quantitated using Image J software.
[0132] .gamma.H2AX Immunofluorescence
[0133] Cells grown on 8-mm glass coverslips were washed three times
with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized
with 0.2% Triton X-100 for 5 min and blocked with 2% BSA for 1 h.
Cells were then incubated with rabbit anti-phospho-histone
.gamma.H2A.X (Ser139) (1:200; Cell Signaling Technology, #9718)
overnight at 4.degree. C., washed, and further incubated with
anti-rabbit secondary antibody, tagged with Alexa Fluor 594 (Life
Technologies, Carlsbad, Calif., USA). For controls, cells were
treated with isotype-matched IgG from the same species. Slides were
mounted using ProLong Gold Antifade Reagent (Life Technologies).
Fluorescence images were captured by confocal laser scanning
microscope. .gamma.H2AX fluorescence was quantitated in 5-6 fields
for each condition using ImageJ (NIH, Bethesda, Md.), and corrected
for the number of nuclei per field (average=14), visualized by DAPI
staining. Data were calculated from three replicate
experiments.
[0134] Discovery of IGFBP-3-Interacting Proteins
[0135] MDA-MB-468 cells were grown to 90% confluence in T75 flasks
in RPMI 1640 medium containing 5% fetal calf serum and 10 .mu.g/mL
bovine insulin, then exposed to 20 .mu.M etoposide, or medium alone
for control cells, for 2 h. Medium was removed, and cells were
washed twice in PBS, then lysed with 1 mL ice-cold RIPA buffer
supplemented with protease and phosphatase inhibitors (as above) at
4.degree. C. for 30 min. After centrifugation to remove insoluble
material, the supernatant was incubated overnight with anti-IGFBP-3
IgG (Fab fraction) conjugated to agarose beads as previously
described (Lin M Z et al., Oncogene, 2014, 33, 85-96). Control
precipitations used agarose beads without antibody. Beads were
pelleted by centrifugation, washed 4 times in ice-cold PBS,
resuspended in 50 .mu.L 0.1% solution of RapiGest SF surfactant
(Waters, Rydalmere, NSW, Australia) in 20 mM Tris-HCl buffer, pH
7.4. After boiling for 5 min to dissociate immunoprecipitated
proteins, supernatants were collected by centrifugation and stored
at -80.degree. C. before analysis. For proteomic analysis,
tris(2-carboxyethyl) phosphine was added to 5 mM final
concentration, samples were heated at 60.degree. C. for 30 min,
then cooled to room temperature. Iodoacetamide was added to 15 mM
and reacted for 30 min in the dark. Trypsin Gold (MS grade;
Promega, Alexandria, NSW, Australia) was added at 1:50 by protein
weight, the solutions were incubated overnight at 37.degree. C.,
and TFA was added to 0.5% final. After 45 min at 37.degree. C.,
samples were immersed in liquid nitrogen to precipitate the
RapiGest, then centrifuged for 10 min, and the supernatants
collected. Samples were fractionated on an UltiMate 3000 nanoLC
(Thermo Scientific) and spotted onto a Bruker MTP 384 AnchorChip
target plate (Bruker, Preston, VIC, Australia) using a Proteineer
fc II fraction collector (Bruker) as described previously (Hunt N
J., J Proteom., 2016, 138, 48-60). MS/MS data were acquired on an
UltrafleXtreme MALDI TOF/TOF mass spectrometer (Bruker) with a
smart beam laser run at 2 kHz, with data processing and peptide
identification performed as previously described (Hunt N J., J
Proteom., 2016, 138, 48-60).
[0136] NONO-IGFBP-3 Binding Assay
[0137] NONO was diluted in 50 mM sodium phosphate, 0.05% BSA, pH
7.4, and incubated 16 h at indicated concentrations in wells of
FLAG (i.e. DDK) antibody plates. All incubations were at 22.degree.
C. in 100 .mu.L of 0.1 M Tris-HCl, 0.05% BSA, pH 7.4 (incubation
buffer) unless noted otherwise. After 4 washes with 250 .mu.L cold
incubation buffer, wells were incubated for 2 h at 22.degree. C.
with recombinant human IGFBP-3 at indicated concentrations in
incubation buffer containing 1% BSA. After 4 washes as above, wells
were incubated 2 h with anti-human IGFBP-3 antiserum R-100 at
1:25,000, washed 4 times, incubated 1 h with goat anti-rabbit
IgG-HRP at 1:20,000, washed 4 times, and incubated 30 min with 100
.mu.L TMB solution. Reactions were stopped by adding 100 .mu.L 1 M
H.sub.2SO.sub.4 and absorbance read at 450 nm.
[0138] DNA End-Joining Assay
[0139] Nuclear extraction and end-joining assay was performed as
previously described (Andrin C et al., J Blot Chem., 2004, 279,
25017-25023; Andrin C et al., Nucleus., 2012, 3, 384-395) with
slight modifications. Briefly, HCC1806 cells were grown in flasks
and treated with inhibitors for 24 h followed by etoposide
treatment for 2 h as described above. After isolation of nuclei by
centrifugation through a buffer containing 300 mM sucrose, the
washed nuclear pellet was extracted into high-salt buffer (20 mM
Hepes, pH 7.5, 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM
MgCl2) for 30 min on ice, and insoluble material was removed by
centrifugation. The soluble nuclear extract was used in the
end-joining assay. Restriction enzymes NheI and EcoRI (New England
Biolabs, Ipswich, Mass., USA) were used to digest a EGFP-C1 plasmid
(Clontech, Mountain View, Calif., USA) to generate a DNA fragment
of 4 kb with non-homologous ends. The linearized plasmid was
separated by 0.8% agarose gel electrophoresis, purified using a DNA
gel extraction kit (Qiagen), and used as the substrate for
end-joining assays. Nuclear extract (2 .mu.g) was mixed with
end-joining assay buffer (7.5 mM Tris pH 8.0, 0.2 mM CaCl.sub.2, 10
mM MgCl.sub.2, 50 mM KCl, 1.2 mM ATP and 0.5 mM DTT) and allowed to
stand for 30 min at 22.degree. C. Repair was initiated by adding
100 ng of prepared linearized DNA and incubated at 25.degree. C.
for 30 min, stopped by the addition of 0.5 M EDTA, 0.5% SDS and 10
mg/mL Proteinase K. DNA bands were separated on a 0.7% agarose gel,
stained with SYBR Gold (Life Technologies), and visualized on a
BioRad ChemiDoc imaging system.
[0140] Generation and Testing of Inhibitory Peptides
[0141] A library of 85 overlapping 12-residue peptides covering the
full-length sequence of mature human IGFBP-3 (264 residues) was
synthesised and purified to at least 80% purity by ChinaPeptides
Co., Shanghai, China. The overlap was nine residues, i.e. residues
1-12, 4-15, . . . 250-261, 253-264. For each peptide, 5 mg
(calculated as approx. 3.79 .mu.mol) was dissolved in 379 .mu.l of
20% acetonitrile in water, to give a concentration of 10 mM. For
screening assays, NONO was bound to each well at 240 ng/100 .mu.l.
After 16 h incubation and washing as described above, the IGFBP-3
peptides, diluted 1:500 to 20 .mu.M in incubation buffer, were
added diluted 1:1 with recombinant IGFBP-3 (25 ng) in a total
volume of 100 .mu.l incubation buffer. The final peptide
concentration was 10 .mu.M and the final IGFBP-3 concentration
approx. 6 nM. After 2 h incubation the IGFBP-3 binding was
determined as described above.
[0142] Epitope Mapping
[0143] To further refine the amino acid residues of IGFBP-3
involved in the interaction between IGFBP-3 and NONO, three
additional derivatives of the peptide
His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser-Pro-Arg-Gly were synthesized
and tested: [0144] (1A):
His-Ala-Lys-Phe-Ala-Asn-Val-Ala-Ser-Pro-Arg-Gly, in which the three
Leu residues were changed to Ala [0145] (2A):
His-Leu-Ala-Phe-Leu-Asn-Val-Leu-Ser-Pro-Ala-Gly, in which the two
basic amino acids Lys and Arg were both changed to Ala [0146] (3A):
His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser, in which the three
carboxyterminal residues were deleted.
[0147] Each peptide was tested for its inhibitory activity in the
cell-free IGFBP-3-NONO binding assay and the cell-based
co-immunoprecipitation assay, and, if found to be inhibitory, was
further evaluated for its effect on .gamma.H2AX immunofluorescence
following etoposide treatment of cells.
[0148] Effects of Peptide on the .gamma.H2AX Response to Etoposide
in Breast Cancer Cells [0149] MDA-MB-468 or HCC1806 human TNBC
cells were preincubated for 24 h with PARP inhibitors olaparib or
veliparib at 0, 1 or 10 .mu.M, without or with 10 .mu.M peptide.
Etoposide (at the indicated final concentration) was added for 1 h,
then cell lysates were harvested in Laemmli buffer, separated by
SDS-PAGE, and blotted for .gamma.H2AX.
[0150] Statistics
[0151] ANOVA with post hoc Fisher's LSD test (SPSS v.22 for Mac;
IBM Corp, Armonk, N.Y., USA) was used for multiple group
comparisons.
Example 1
NONO Interacts with IGFBP-3
[0152] An unbiased proteomic screen for proteins that interact with
IGFBP-3 2 h after etoposide treatment was carried out. Examination
by LC-MALDI-TOF/TOF mass spectrometry of proteins co-precipitating
with IGFBP-3 from whole cell lysates consistently revealed NONO as
a putative IGFBP-3 binding partner. Unique peptides for the NONO
protein, identified by mass spectrometry from
IGFBP-3-coimmunoprecipitation (coIP) experiment are shown in Table
2.
TABLE-US-00002 TABLE 2 Unique NONO peptides identified by
LC-MALDI-TOF/TOF from IGFBP-3 co-immunoprecipitation experiments
.DELTA. m/z Accession Range m/z meas. Mr calc. [ppm] Sequence
Modifications NONO_HUMAN 143-153 1248.6073 1427.6081 -6.49
R.FACHSASLTVR.N Carbamidomethyl: 3 NONO_HUMAN 191-202 1231.6920
1230.6721 10.25 K.GIVEFSGKPAAR.K NONO_HUMAN 357-365 1263.5989
1262.5972 -4.45 R.RQQEEMMRR.Q NONO_HUMAN 365-378 1636.8313
1635.8118 7.48 R.RQQEGFKGTFPDAR.E NONO_HUMAN 366-378 1480.7236
1479.7106 3.84 R.QQEGFKGTFPDAR.E NONO_HUMAN 384-398 1538.6691
1537.6622 -0.23 R.MGQMAMGGAMGINNR.G NONO_HUMAN 435-456 2163.0680
2162.0579 1.31 R.FGQAATMEGIGAIGGTPPAFNR.A NONO_HUMAN 435-468
3372.8220 3371.6833 38.96 R.FGQAATMEGIGAIGGTPPAFNRAA PGAEFAPNKR.R
NONO_HUMAN 457-468 1228.8834 1227.6360 18.35 R.AAPGAEFAPNKR.R
NONO_HUMAN 457-469 1384.7644 1383.7371 14.40 R.AAPGAEFAPNKRR.R
[0153] The interaction, and its stimulation by chemotherapy
treatment, were confirmed by coIP and western blotting (FIG. 1),
and by proximity ligation assay (PLA, FIG. 2). FIG. 1a shows
western blots of whole cell lysates from MDA-MB-468 cells treated
with etoposide for 0 to 4 h, after immunoprecipitation using
agarose-immobilized anti-human IGFBP-3 IgG (Fab fragment) or
control, non-immune agarose beads. CoIP of NONO typically peaked
after 2 h exposure to etoposide, although the time-course was
variable among experiments, with earlier (1 h) or later (4 h) peaks
seen in some experiments. This variability may be related to the
passage number of the cells, with the peak time tending to increase
with extended passages after thawing. Weak bands for both antigens
were also seen in some control IPs.
[0154] When IGFBP-3 was downregulated transiently in MDA-MB-468
cells by siRNA, the amount of NONO detectable after IP with
anti-human IGFBP-3, 2 h after etoposide treatment, was greatly
reduced compared to that from cells treated with control
non-silencing siRNA (FIG. 1b) thus providing further support that
NONO was precipitating in a complex containing IGFBP-3.
Immunoprecipitated IGFBP-3 was detected as a diffuse band around 40
kDa (known to be a mixture of glycosylation isoforms) plus a weak
band, probably proteolyzed IGFBP-3, below 30 kDa. An increase in
IGFBP-3-associated NONO after etoposide treatment was similarly
observed in isolated nuclear extracts rather than whole cell
lysates (FIG. 1c). Similar to MDA-MB-468 cells, IGFBP-3-associated
NONO also increased in HCC1806 basal-like breast cancer cells in
response to etoposide, typically peaking 1-2 h after etoposide
treatment (FIG. 1d). A representative image of immunoprecipitated
IGFBP-3, measured in most coIP experiments, is also shown in FIG.
1d. In FIG. 1e, the association of NONO with IGFBP-3 in HCC1806
cells is quantitated for 5 experiments, the broad peaks
representing the somewhat variable time-courses. IGFBP-3
interaction with NONO was also demonstrated in a cell-free system.
This was examined using a direct binding assay in which IGFBP-3
bound to immobilized NONO was detected in an ELISA format. FIG. 1f
shows dose-response curves for a fixed IGFBP-3 concentration (10
ng/100 .mu.L; approx. 2.5 nM) bound to increasing concentrations of
immobilized NONO, and for increasing IGFBP-3 concentrations bound
to a fixed amount of NONO (25 ng/100 .mu.L; approx. 4.6 nM). The
NONO-IGFBP-3 interaction appears dose dependent and saturable,
consistent with NONO forming a specific protein--protein
interaction with IGFBP-3.
[0155] FIG. 2 confirms the association of NONO with IGFBP-3 in
breast cancer cells by proximity ligation assay (PLA). Biomolecular
interaction between IGFBP-3 and NONO was minimal before etoposide
treatment, typically peaking 2 h after exposure to 20 .mu.M
etoposide and decreasing again at 4 h. In control PLA experiments,
in which either detection antibody was omitted, no signal was
observed (not shown). These independent approaches confirm that
NONO forms transient nuclear complexes with IGFBP-3 in basal-like
TNBC cells treated with etoposide.
Example 2
The Effects of PARP Inhibition on IGFBP-3 Interaction with NONO
[0156] Since NONO recruitment to DNA damage sites is reported to be
PARP-dependent (Krietsch J., Nucl Acids Res., 2012, 40,
10287-10301), we examined the effect of PARP inhibition on the
interaction between IGFBP-3 and NONO. FIG. 3a shows in MDA-MB-468
cells that IGFBP-3 complexes with NONO, determined by
immunoblotting after coIP, were abolished if cells were
preincubated with the PARP1 and PARP2 inhibitor veliparib (20
.mu.M) for 24 h prior to exposure to etoposide. Data for 3
experiments in MDA-MB-468 cells shown in FIG. 3b for IGFBP-3-NONO
interactions. A similar inhibitory effect was seen after
preincubation with a second PARP inhibitor, olaparib at 10 .mu.M
(FIG. 3c). The inhibitory effect of veliparib on complex formation
was confirmed by PLA in both MDA-MB-468 and HCC1806 cells (FIG.
3d), showing the increase in IGFBP-3-NONO complexes 2 h after
etoposide treatment was abolished by preincubation with 20 .mu.M
veliparib. FIG. 3e shows the quantitation of 3 replicate
experiments in MDA-MB-468 cells, with the effect of veliparib
highly significant by ANOVA (P<0.001). Therefore, the formation
of EGFR-dependent complexes between IGFBP-3 and NONO in basal-like
TNBC cell lines exposed to DNA-damaging chemotherapy requires PARP
activity.
[0157] Consistent with the above, DNA repair activity in TNBC cell
lines was inhibited by PARP inhibitors. As shown in FIG. 4a, c,
treatment of either MDA-MB-468 or HCC1806 cells with etoposide for
1 h (T0) caused a significant increase in foci of histone H2AX
phosphorylated on serine 139 (.gamma.H2AX), which accumulates at
sites of DNA double-strand breaks. This signal had substantially
declined after 4 h (T4), consistent with DNA repair over this
period. The addition of either PARP inhibitor, olaparib or
veliparib, significantly prevented the loss of .gamma.H2AX signal,
indicating that both drugs were inhibitory to DSB repair in these
cell lines, neither of which has a mutation in BRCA1 or BRCA2. Data
for both cell lines are quantitated in FIG. 4b, d. Etoposide
treatment also increased activity in a direct DNA end-joining assay
using nuclear extracts from treated cells (FIG. 4e). In extracts
from cells treated with either PARP inhibitor, end joining activity
was inhibited by approximately 50% in both MDA-MB-468 and HCC1806
cells, as shown quantitatively in FIG. 4f.
Example 3
Inhibition of NONO-IGFBP-3 Interaction
[0158] Peptide #66 consistently inhibited IGFBP-3 binding to NONO
in the screening assay. This peptide has the sequence HLKFLNVLSPRG
(i.e. His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser-Pro-Arg-Gly). FIG. 5a
shows results from a NONO-IGFBP-3 binding assay comparing peptides
#64 to 67 to no added peptide. FIG. 5b shows the mean inhibitory
effect of peptide #66 on the NONO-IGFBP-3 interaction from 3
assays, indicated by the lower absorbance value.
Example 4
Sustained DNA Damage
[0159] FIG. 5c and FIG. 5d shows .gamma.H2AX signals in two BRCA
wild-type breast cancer cell lines MDA-MB-468 and HCC1806,
respectively, exposed to 25 .mu.M etoposide, after 16 h incubation
with the indicated peptides #64 to #66 at 10 .mu.M, or no peptide.
In this experiment, peptides #65 (TLNHLKFLNVLS) and #66
(HLKFLNVLSPRG) with overlapping residues HLKFLNVLS (i.e.
His-Leu-Lys-Phe-Leu-Asn-Val-Leu-Ser) caused a sustained .gamma.H2AX
signal compared to no peptide or peptide #64 (MEDTLNHLKFLN) in
which a low .gamma.H2AX signal was seen. An elevated .gamma.H2AX
signal is indicative of sustained DNA damage (i.e. inhibition of
DNA repair) compared to the control cells. Peptide #66 was also
evaluated in the presence of PARP inhibitors, veliparib (ABT-888)
and olaparib (Lynparza). In FIG. 5e and FIG. 5f, the .gamma.H2AX
signal in the absence of peptide #66 is expressed as 100%, and bars
represent the ratio of .gamma.H2AX signal in the presence of the
peptide, to that in its absence. Preincubation with either PARP
inhibitor in the presence of peptide #66 increased the .gamma.H2AX
signal by almost 2-fold at 10 .mu.M of PARP inhibitors (FIG. 5e:
veliparib; FIG. 5f: olaparib), suggesting a combination effect
between the PARP inhibitor and the inhibitor of NONO-IGFBP-3
interaction i.e. peptide #66. These results demonstrate that
inhibition of NONO-IGFBP-3 interaction can act in conjunction with
PARP inhibition as an effective means to inhibit DNA damage repair
following exposure to a DNA-damaging chemotherapy drug such as
etoposide.
Example 5
Peptide #66 Inhibits the Complex Formation Between IGFBP-3 and
NONO/SFPQ in TNBC Cell Lines
[0160] Using TNBC cells lines as described above, MDA-MB-468 and
HCC1806, it was demonstrated that peptide #66 inhibited the complex
formation between IGFBP-3 and NONO/SFPQ (FIG. 6). MDA-MB-468
basal-like TNBC cells were incubated for 1 h without (control) or
with 10 .mu.M of peptide #66 prior to exposure to 20 .mu.M
etoposide (Etop) for the indicated times (0, 1, 2, or 4 h), after
which NONO and SFPQ were precipitated from cell lysates by
anti-IGFBP-3 antiserum (Fab fraction) coupled to agarose beads
(FIG. 6a). The samples were blotted for NONO or SFPQ after
fractionation by SDS-PAGE. HCC1806 basal-like TNBC cells were
treated and analysed as described in above, except that the
concentration of peptide #66 was 20 .mu.M (FIG. 6b). Preincubation
for 1 h with peptide #66 inhibited the subsequent formation of
complexes between IGFBP-3 and NONO or SFPQ after etoposide
exposure, as indicated by the decreased density of bands detected
by immunoblotting (FIG. 6). The inhibition of complex formation
after etoposide exposure indicates that the repair of DNA damage,
caused by the chemotherapy agent (Etop), is impaired in the
presence of peptide #66.
Example 6
Peptide #66 Makes Breast Cancer Cells More Responsive to the Effect
of a PARPi
[0161] In a 14-day colony formation or clonogenic survival assay,
it was shown that the survival of HCC1806 breast cancer cells for
12 days after a 2-day exposure to a low concentration of etoposide
(100 nM), was inhibited maximally by a combination of a PARPi
(veliparib) and peptide #66. HCC1806 cells (500 cells/well) were
plated in 6-well plates for 24 h prior to being treated with or
without 5 .mu.M of PARPi (veliparib) for a further 24 h. Cells were
then exposed to 20 .mu.M of peptide #66 (or not, as indicated) for
1 h, followed by 100 nM of etoposide treatment (or not, as
indicated) for 48 h, after which the medium was replaced with fresh
medium. Colony formation was observed for a further 12 days during
which cells were refreshed with new media every 3 days. Colonies
were washed with PBS and stained using 0.5% Crystal Violet (Sigma
Aldrich) in 20% methanol for 30 min prior to rinsing with water.
Colonies, defined as clusters of at least 30 cells, were imaged and
counted with an AID vSpot Spectrum imager (AutoImmun Diagnostika
GmbH, Strassberg, Germany).
[0162] The 14-day colony formation or clonogenic survival assay
measures the ability of cells to survive 2 days of
chemotherapy-induced DNA damage, and form colonies of at least 30
cells over the next 12 days. The etoposide concentration used was
very low (100 nM), so that only minor cell death would occur under
control conditions. The purpose was to find conditions under which
the cells become more sensitive to this low dose of chemotherapy.
In the absence of etoposide, the PARP inhibitor veliparib, at the
concentration used (5 inhibited cell survival by about one-third,
and peptide #66 had no additional effect (FIG. 7). In contrast, in
cells exposed to etoposide, the PARP inhibitor again inhibited cell
survival by about one-third, but the addition of peptide #66 caused
highly significant further inhibition of cell survival (colony
formation). This experiment provides a proof of principle that
peptide #66 can act in combination with PARP inhibition to enhance
the ability of chemotherapy to inhibit breast cancer cell
survival.
Example 7
Peptide #66 Rapidly Enters the Nucleus of Breast Cancer Cells
[0163] Fluorescently-labelled peptide #66 was used to demonstrate
that the peptide can directly diffuse into the cell nuclei. The
peptide was synthesised with the fluorescent dye 5-TAMRA
(5-carboxytetramethyl-rhodamine) covalently bound at its
amino-terminus. Since under some circumstances the fixation of
cells prior to imaging may introduce artefacts, experiments were
performed to detect the localisation of the peptide both with and
without fixation of the cells. Fixed cell imaging (FIG. 8a):
HCC1806 cells were grown on 8 mm glass coverslips; 10 .mu.M of
peptide #66 labelled with 5-TAMRA was mixed with complete growth
medium containing serum, and directly added to the cells. At the
indicated time points, cells were washed three times with PBS,
fixed with 4% paraformaldehyde for 15 min, washed three times and
mounted on slides using ProLong Gold Antifade Reagent containing
the nuclear stain DAPI (Life Technologies). Confocal fluorescence
images were captured with a Leica TCS SP5 confocal laser scanning
microscope (Leica Microsystems, Wetzlar, Germany). Live cell
imaging (FIG. 8b): HCC1806 cells were plated in an 8-well chamber
slide (Nunc Lab-Tek, Sigma Aldrich); 0.1 mg/mL Hoechst nuclear
stain (Life Technologies) was added to the cells for 15 mins and
imaged (0 min time point) using Leica TCS SP5 confocal microscope.
The medium was then changed to complete growth medium containing
serum plus 10 .mu.M of peptide #66 labelled with 5-TAMRA. Images
were taken at the times indicated.
[0164] Fixed cell imaging showed rather diffuse green staining
associated with the cells after 30 min, strongly associated with
cell nuclei as indicated by the cyan colour of the merged nuclei
(blue) and labelled peptide (green) images. The staining was less
intense after 60 min, but the nuclear localisation of the labelled
peptide remained very clear. The confocal microscopy images are
taken at the plane of the center of cell nuclei, indicating that
dye associated with the nuclei is likely to be intranuclear. In
live cell imaging, the labelled peptide appeared less diffuse, as
indicated by the punctate red staining. In this experiment the
labelled peptide was associated with cell nuclei at 40 and 60 min,
and even more so after 90 min. As with the fixed cell imaging,
these images are taken at the plane of the center of cell nuclei,
indicating that the labelled peptide associated with nuclei is
likely to be intranuclear. These experiments indicate that there is
rapid nuclear uptake of peptide #66 by these breast cancer cells,
consistent with the data that a 1-h preincubation of cells with
peptide #66 is sufficient to inhibit the formation of complexes
between IGFBP-3 and NONO/SFPQ as shown in FIG. 6.
Example 8
Peptide #66 Inhibits the Complex Formation Between IGFBP-3 and
NONO/SFPQ in Glioblastoma Cell Lines
[0165] Glioblastoma represents another type of IGFBP-3 expressing
cancer. It was demonstrated that in 2 glioblastoma cell lines, A172
and M059K, etoposide stimulated the formation of complexes between
IGFBP-3 and NONO/SFPQ as seen in breast cancer cells (FIG. 9a). The
cell line, M059K, was used to test the effects of peptide #66 on
these complexes (FIG. 9b). The human glioblastoma cell line A172
was maintained in DMEM with 10% fetal calf serum, and M059K cells
were maintained in DMEM/F12 containing 10% fetal calf serum under
standard conditions. A172 or M059K human glioblastoma cells were
exposed to 20 .mu.M of etoposide (Etop) for the indicated times (0,
1, 2, or 4 h), after which NONO and SFPQ were precipitated from
cell lysates by anti-IGFBP-3 antiserum (Fab fraction) coupled to
agarose beads. The samples were blotted for NONO or SFPQ after
fractionation by SDS-PAGE. For the experiment shown in FIG. 9b,
M059K human glioblastoma cells were incubated for 1 h without
(control) or with 25 .mu.M of peptide #66 prior to exposure to 20
.mu.M of etoposide (Etop) for the indicated times (0, 1, 2, or 4
h), after which NONO and SFPQ were precipitated from cell lysates
by anti-IGFBP-3 antiserum (Fab fraction) coupled to agarose beads.
The samples were blotted for NONO or SFPQ after fractionation by
SDS-PAGE. FIG. 9a demonstrates that human glioblastoma cell lines
form the same etoposide-stimulated complexes containing IGFBP-3 and
NONO/SFPQ as previously observed to form in breast cancer cell
lines. FIG. 9b shows that the formation of these complexes is
inhibited by exposure of the cells to peptide #66, suggesting that
the peptide is likely to inhibit DNA damage repair and therefore
have an effect on glioblastoma cell survival as observed for breast
cancer cells.
* * * * *